DE68923801T2 - METHOD FOR PRODUCING GANGLIOSIDE GM1. - Google Patents

Description

This invention relates to a process for the production of ganglioside GM1.

The term "ganglioside" is a generic term for a class of sialic acid-containing sphingoglycolipids and describes a substance that occurs in the brain of higher animals, particularly in the nervous system. In the living organism, gangliosides are not only a component of nerve function and the recognition, differentiation, proliferation, carcinogenesis, aging, etc. of cells, but also with regard to "cell sociology" they are responsible for the receptor function of cytotoxins, for example cholera toxin and botulinum toxin, peptide hormones, for example thyroid hormone, interferons, etc. It is assumed that the gangliosides contribute to negative charges on cell surfaces. It is also described that the gangliosides extracted from bovine brain promote the regeneration of damaged peripheral nerves and are useful for the treatment of alcoholic polyneuritis, diabetic neuropathy, etc. (Iyaku to Yakugaku 13(6) (1985), 1407-1412, Practice 4(4) (1987), 452-456, and Iyaku Journal 22(7) (1986), pp. 55-62).

As described above, gangliosides are important substances with various functions in the living organism. Therefore, it is desirable to establish a method for the purification of the individual gangliosides. Up to now, attempts have mostly been made to isolate a certain ganglioside by different chromatographic methods. However, since the starting material, ie the ganglioside components extracted from bovine brain or the like, is a mixture containing only traces of different gangliosides, it is difficult to obtain the desired products even if complex processes are used and suitable purification methods have not yet been found. Chemical synthesis of gangliosides has been attempted, but it is complicated and side reactions are likely to occur, producing by-products that are difficult to remove.

Neuraminidase, on the other hand, is an enzyme that does not act directly on gangliosides. It is also only reported that asialoganglioside GA1 is formed from ganglioside GM1 in the presence of detergents, such as cholates (Masaki Saito, Taisha 16 (1977), 761).

One aspect of the invention is to provide a process for obtaining a particular ganglioside, i.e., ganglioside GM1, from gangliosides.

Another aspect of the invention is to provide a process for producing ganglioside GM1 with high purity and high yield.

In particular, the present invention provides a process for the preparation of ganglioside GM1, which is characterized in that neuraminidase isozyme S is allowed to act on gangliosides to obtain ganglioside GM1.

Through intensive research, it has been found that neuraminidase derived from bacteria of the genus Arthrobacter is a new isozyme and that by acting on gangliosides, ganglioside GM1 can be selectively obtained with high purity and in good yield. No by-products are produced in the process of the invention and therefore the obtained ganglioside GM1 can be easily purified. The present invention also has the advantage that ganglioside GM1 can be produced using this enzyme in the absence of a detergent.

The neuraminidase isozyme S (hereinafter referred to as "isozyme S") used in this invention has the properties described below:

(1) Effect: selective production of ganglioside GM1 from gangliosides

(2) Molecular weight: about 52 000 Dalton

Molecular weight was determined by gel filtration chromatography and SDS-PAGE electrophoresis as described below.

[Gel filtration chromatography]

The gel filtration procedure was carried out using Sephadex G150 (Pharmacia). 50 mM phosphate buffer was used as eluent.

[SDS-PAGE electrophoresis]

Electrophoresis was performed using an SDS-polyacrylamide gel according to the method of U. K. Laemmli (Nature 227 (1970), 680).

(3) PH optimum: 3.8 to 4.4 (using bovine brain gangliosides as substrate)

(4) Stable pH range: 3.5 to 10.0

(5) Optimum temperature: 45 to 55ºC

(6) Heat stability: 60ºC or below.

On the other hand, neuraminidase derived from Arthrobacter ureafaciens was classified into type I and type II. Their physicochemical properties are described below in Table I (Y. Uchida et al., J. Biochem. 86 (1979) , 425) Table I Molecular weight (gel filtration method) pH optimum Stable pH range Temperature optimum Heat stability

The above description shows that Isozym S is characterized by its physico-chemical properties, for example molecular weight, pH optimum, stable pH range, temperature optimum and heat stability, differs from the usual neuraminidase and that they are therefore enzymes consisting of different types of molecules.

The activity of isozyme S is determined as for neuraminidase, for example according to the thiobarbituric acid method described in J. Biol. Chem. 234 (1959), 1971. One unit of isozyme S is defined as the amount of enzyme which liberates 1 µmol of N-acetylneuraminic acid per minute at 37ºC.

Isozyme S can be obtained from the culture obtained by culturing a bacterium of the genus Arthrobacter.

Known bacteria can be used as bacteria of the genus Arthrobacter. Examples are strains of Arthrobacter ureafaciens, for example Arthrobacter ureafaciens M1057 (FERM BP-1391).

Incubation of the bacteria of the genus Arthrobacter can be carried out using common liquid or solid media, but it is usually advantageous to use a liquid medium. For the production of the desired enzyme, incubation is preferably carried out with shaking or with aeration and stirring. There is no particular limitation on the choice of media that can be used for the incubation described above, and various media containing nutrients and similar substances commonly used for incubation of microorganisms can be used. Examples of nutrients are sugars, for example glucose, fructose, lactose, invert sugar, saccharified starch solution, sialic acid, sorbitol and glycerin, organic acids, for example pyruvic acid, malic acid and succinic acid, nitrogen sources, for example peptides, meat extract, yeast extract, casamino acids, urea, ammonium salts and nitrate salts, inorganic phosphorus, magnesium, potassium, sodium salts etc., trace elements, for example boron, copper, tod, iron, manganese, zinc, cobalt and molybdenum and growth factors present in trace amounts, for example vitamins. Other examples of the media described above are natural or semi-synthetic media containing an extract or exudates from animal tissues. Examples of these media are commercially available under the name "Todd Hewitt Broth Medium", "Brain Heart Infusion Medium", etc.

The incubation was usually carried out at a temperature of about 20 to about 40°C, preferably about 25 to about 30°C, and was terminated after about 10 to about 70 hours.

Isozyme S can be obtained from the culture solution of bacteria of the genus Arthrobacter in the usual way. The bacteria are, for example, isolated and removed from the culture solution, and the resulting supernatant is purified, for example, by ammonium sulfate fractionation and affinity chromatography and, if necessary, by further purification steps, for example ion exchange chromatography, chromatographic focusing and gel filtration.

By allowing the resulting isozyme S to act on gangliosides, ganglioside GM1 is selectively obtained. This enzymatic reaction is carried out by adding isozyme S to the starting solution, which contains gangliosides and buffer and, if necessary, sterilized water.

For the above-described enzymatic reaction, known gangliosides, for example, at least one of gangliosides GD1a, GD1b, GT1a, GT1b and GQ1b or a ganglioside mixture extracted from bovine brain, etc., can be used as starting materials of ganglioside GM1 without any particular limitation. The amount of gangliosides is not particularly limited and is usually not more than 50 mg, preferably about 0.1 to about 3 mg, per ml of starting solution. Usual buffers having a pH of about 3 to about 6 can be used, and examples thereof are a buffer containing about 10 to about 200 mM acetate (pH of 3.5 to 5.0), McIlvaine buffer (pH 3.5 to 5.5) etc. The amount of isozyme S is not limited to a specific amount and can be varied within a wide range. It is usually not less than 10 mU, preferably about 0.1 to about 10 U, per ml of starting solution. The temperature and pH of the reaction are arbitrary as long as the enzyme is active, and the temperature can usually be about 20 to about 50ºC and the pH about 3 to about 6. The appropriate reaction time is selected depending on the concentration of the substrate, the reaction temperature, etc. The reaction time is, for example, about 30 minutes to 24 hours at 37ºC.

Conventional purification methods can be used to purify ganglioside GM1 from the reaction mixture. For example, purification can be carried out by subjecting the reaction mixture to solvent extraction and then carrying out concentration, desalination, freeze-drying, etc.

Examples

The present invention will be described in more detail with reference to the following Reference Example and Examples.

Reference example 1

A medium (pH 7.0) containing 5.0 g of lactose, 2.0 g of diammonium hydrogen phosphate, 3.0 g of sodium chloride, 1.0 g of dipotassium hydrogen phosphate, 0.1 g of magnesium sulfate and 1,000 ml of deionized water was placed in a 21 Sakaguchi bottle. The medium was then sterilized by heating in an autoclave.

Arthrobacter ureafaciens M1057 was inoculated into this medium, after which the culture was shaken at 28ºC for 24 hours. The resulting culture solution was centrifuged (10,000 rpm, 10 minutes) to obtain a culture supernatant.

Ammonium sulfate was then added to the culture supernatant and the fraction obtained from a supernatant saturated to 80% with ammonium sulfate was collected. The precipitate was dissolved in a small amount of water and then dialyzed against 10 mM acetate buffer (pH 4.5). The dialysate was passed through the column filled with the gel prepared by adding epichlorohydrin to a 2 N sodium hydroxide solution containing soluble starch and sialic acid, in which sialic acid was a ligand. The adsorbed neuraminidase was then eluted with 100 mM acetate buffer (pH 4.5).

The neuraminidase-active fractions were salted out with ammonium sulfate and dialyzed against deionized water. The dialysate was separated into three components by chromatographic focusing. Chromatographic focusing was carried out as described below. The dialysate was passed through a column of polybuffer exchanger equilibrated with 25 mM imidazole-HCl (pH 7.4) and eluted with polybuffer 74 (Pharmacia) adjusted to pH 3.8.

The three components obtained were separately salted out with ammonium sulfate and dissolved in 100 mM phosphate buffer (pH 7.0). The solution was subjected to gel filtration with Ultrogel AcA44 (I. B. F. Biotechnics) to obtain 4 components. Isozyme S (650 units) was obtained from one of these 4 components.

The enzyme activity of Isozyme S was determined according to the thiobarbituric acid method as described below.

0.1 ml of enzyme solution, 0.05 ml of 0.4% N-acetylneuraminyllactose solution and 0.05 ml of 0.2 M acetate buffer (pH 5.0) were incubated for 10 minutes at 37ºC. The amount of N-acetylneuraminic acid released was determined by the thiobarbituric acid method. One unit of isozyme S was defined as the amount of enzyme that released 1 µmol of N-acetylneuraminic acid per minute at 37ºC.

example 1

1 ml of 0.4% ganglioside GT1b solution, 1 ml of 40 mM acetate buffer (pH 4.0) and 1 ml of distilled water were mixed, and 1 ml of an aqueous solution of isozyme S (5 U/ml) was added to the mixture. The mixture was incubated at 37ºC for 1 hour and 1/10 volume of chloroform was added to terminate the enzyme reaction. Ganglioside GM1 was extracted from the reaction solution using a mixture of chloroform and methanol (2:1) and then freeze-dried to obtain 2.4 mg of ganglioside GM1 as a white powder.

Analysis by thin layer chromatography under the conditions described below gave a single spot for the white powder obtained above with the same Rf value (0.48) as an authentic sample of ganglioside GM1 (purity: at least 99%, honing). Thus, it was confirmed that the product was ganglioside GM1 having a purity equal to or greater than that of the authentic sample.

[Thin layer chromatography conditions

Thin layer plate: High performance TLC (HPTLC)

No. 5641 (Merck)

Developer: Mixture of chloroform, methanol and 0.02 % CaCl₂ (60:35:8)

Colorant: Resorcinol-hydrochloric acid reagent

Example 2

1 ml of 0.2% ganglioside GD1b solution, 1 ml of 40 mM acetate buffer (pH 4.0) and 1 ml of sterilized water were mixed, and 1 ml of an aqueous solution of isozyme S (3 U/ml) was added to the mixture, after which it was incubated at 37ºC for 5 hours. Chloroform (1/10) was added to the reaction solution to terminate the enzyme reaction. Ganglioside GM1 was extracted from the reaction solution with a mixture of chloroform and methanol (2:1) and then freeze-dried to obtain 1.5 mg of ganglioside GM1 as a white powder. The preparation of ganglioside GM1 was confirmed in the same manner as in Example 1.

Example 3

The reaction and purification were carried out in the same manner as in Example 2, except that 1 ml of a solution of a ganglioside mixture obtained by extraction from bovine brain (containing 10 mg of gangliosides) was used, whereby about 6.2 mg of ganglioside GM1 was obtained as a white powder. The production of ganglioside GM1 was confirmed in the same manner as in Example 1.

Claims (12)

1. Process for the preparation of a ganglioside GM1, characterized in that, to obtain the ganglioside GM1, neuraminidase isozyme S is allowed to act on gangliosides, whereby the neuraminidase isozyme S is obtained from a culture of a bacterium of the genus Arthrobacter and has the following physico-chemical properties: Action: selective production of the ganglioside GM1 from gangliosides Molecular weight: approximately 52,000 daltons (according to gel filtration chromatography and SDS-PAGE) pH optimum: 3.8 - 4.4 (where bovine brain gangliosides are used as substrate) Heat stability: 60ºC or below. 2. The method of claim 1, wherein the bacterium of the genus Arthrobacter is Arthrobacter ureafaciens. 3. The method of claim 1 or 2, wherein Arthrobacter ureafaciens is Arthrobacter ureafaciens M1057 (FERM BP- 1391). 4. The method according to any one of claims 1 to 3, wherein the gangliosides are at least one of the gangliosides GD1a, GD1b, GT1a, GT1b and GQ1b or a mixture of gangliosides extracted from bovine brain. 5. The method according to any one of claims 1 to 4, wherein the neuraminidase isozyme S is added to a starting solution containing the gangliosides and a buffer to carry out a reaction. 6. The method of claim 5, wherein the starting solution further contains sterilized water. 7. The method according to claim 5, wherein the amount of gangliosides is not more than 50 mg per ml of starting solution. 8. The method of claim 7, wherein the amount of gangliosides is about 0.1 to about 3 mg per ml of starting solution. 9. The method according to claim 5, wherein the amount of neuraminidase isozyme S is not less than 10 mU per ml of starting solution. 10. The method of claim 9, wherein the amount of neuraminidase isozyme S is about 0.1 to about 10 U per ml starting solution. 11. A process according to any one of claims 1 to 10, wherein the reaction is carried out at a temperature of about 20 to about 50°C. 12. The method of any one of claims 1 to 11, wherein the reaction is carried out at a pH of about 3 to about 6.